Development and validation of UHPLC-MS/MS method for simultaneous quantification of escitalopram and its major metabolites in human plasma and its application in depressed patients

Escitalopram, one of the Selective Serotonin Reuptake Inhibitors (SSRIs), has been widely used in the patients with major depression. In this study, a simple, sensitive and rapid method was established and validated for simultaneous quantification of Escitalopram (S-CT), desmethyl escitalopram (S-DCT), didemethyl escitalopram (S-DDCT) and escitalopram N-Oxide (S-NOCT) in human plasma by ultra-high performance liquid chromatography-tandem with mass spectrometry (UHPLC-MS/MS). Analytes were extracted from plasma by utilizing protein precipitation and then separated on a Hypersil GOLD C18 column (50 mm x 2.1 mm, 1.9 mu m). The mobile phase was water: acetonitrile (70:30, v/v) with 0.25% formic acid at a flow-rate of 0.3 mL/min, within a 5 min run time. The mass analysis used positive electro-spray ionization (ESI) in selection reaction monitoring (SRM). The calibration ranges of the analytes were: S-CT: 2.0-200.0 ng/mL, S-DCT: 1.0-100.0 ng/ mL, S-DDCT: 0.5-50.0 ng/mL, S-NOCT: 0.2-20.0 ng/mL. The method has been fully validated for selectivity, linearity, accuracy, precision, matrix effect, recovery, stability and carry over and all the results met the admissible limits according to the the US Food and Drug Administration guidelines. Mean plasma concentration (ng/mL) of S-CT, S-DCT, S-DDCT and S-NOCT in 93 depressed patients were 51.10 +/- 45.73, 10.32 +/- 15.25, 1.53 +/- 1.79 and 0.87 +/- 0.94, respectively. it is the first time that a UHPLC-MS/MS method for simultaneous quantification of S-CT and its 3 metabolites in human plasma was established and validated.